A. Background
Provided herein is a new manufacturing process for the production of pharmaceutical grade DNA. Current state of the art techniques for the purification of plasmid DNA rely upon the use of laboratory scale centrifugation, extraction with toxic organic solvents, and the use of animal-derived enzymes (lysozyme, RNase, Proteinase K). Final purification of plasmid DNA from a lysate is accomplished using methods that may include laboratory scale ultra-centrifugation, preparative work gel electrophoresis, and research installation chromatography. None of these techniques are suitable for the large-scale manufacturing of pharmaceutical-grade plasmid DNA. The drawbacks of current techniques are described below.
Current laboratory methods are not amenable to manufacturing plasmid DNA. There are two widely used laboratory methods for the preparation of a crude lysate enriched with plasmid DNA: the boiling method and the alkaline lysis method. Both commonly utilize chicken egg-white lysozyme to break up the bacterial cell wall. Laboratory scale centrifugation is often implemented to separate cellular debris from the crude lysate. Pancreatic RNase is frequently employed to reduce host-derived RNA, which accounts for approximately 75% of the nucleic acid in the crude lysate. Organic extraction with phenol:chloroform:isoamyl alcohol or a variation of this mixture is typically used to reduce contaminating proteins. At this point the crude lysate still contains substantial contaminating host chromosomal DNA. Further treatment is necessary.
The method described above for obtaining a partially purified DNA from a crude lysate is not an optimal protocol for the manufacture of pharmaceutical grade DNA. Animal derived enzymes, such as lysozyme and RNase, present a problem. By virtue of being animal derived, they may introduce viral contamination into the final plasmid product. Organic solvents are also problematic. These chemicals are highly toxic and accordingly must be eliminated in the final dosage form if the product is intended to be a pharmaceutical. Moreover, the solvents add significant expense to the method in terms of not only storage, safe use and disposal of hazardous waste but also validation of their removal.
The plasmid DNA isolated from the crude lysate is further purified most often by cesium chloride/ethidium bromide (CsCl/EtBr) equilibrium ultra-centrifugation. Due to density differences created by the different binding capacities of EtBr to covalently closed circular plasmid DNA, RNA and chromosomal DNA, these three different nucleic acids can be resolved into enriched fractions by CsCl gradient ultra-centrifugation.
CsCl/ EtBr gradient centrifugation is also undesirable as a method for the production of pharmaceutically acceptable DNA. It is not an economically scalable technique for the manufacture of DNA. Also, EtBr is a highly toxic, mutagenic and teratogenic reagent whose presence would not be tolerated, even at trace levels, in a pharmaceutical product and presents significant problems of safe disposal.
There are variations of the methodology described above where the crude lysate is treated with pancreatic RNase followed by an alkali/detergent treatment to reduce chromosomal DNA. An organic extraction with phenol:chloroform is followed by precipitation of DNA by ethanol, re-suspension and a polyethylene glycol (PEG) precipitation of DNA. Again, this is a time-consuming, laboratory-scale methodology not amenable to pharmaceutical manufacturing. It makes use of animal derived enzymes, toxic solvents and reagents not generally recognized as safe (GRAS) by the Food and Drug Administration (FDA).
The new process disclosed herein is suitable to manufacture pharmaceutical-grade DNA for such uses as gene therapy. This process is capable of separating various forms of plasmid DNA including supercoiled, relaxed and concatemers. The DNA produced by this manufacturing process is essentially free or contains only trace levels of host derived contaminants such as proteins, lipids, carbohydrates, endotoxins, chromosomal DNA, and RNA. It is manufactured using no animal- or otherwise-derived enzymes. The purification is accomplished using only reagents generally recognized as safe by the FDA. The manufacturing method of the invention is composed of a novel sequence of unit operations scalable to large quantities of DNA (milligrams, grams, kilograms) and is substantially more economical than current methods. Finally, the sequence of unit operations combined in this manufacturing process is complete including sterile fill of product DNA into appropriate vials. These attributes clearly distinguish the manufacturing process described here from current state of the art methods and make it especially well suited for the manufacture of pharmaceutical grade DNA.